Cell therapy holds significant potential for the reconstruction and regeneration of craniofacial defects and deformities. One of the most important aspects of cell therapy is the identification of the most appropriate cell source for isolation of cells. Historically, mesenchymal stem cells (MSCs) derived from bone marrow have demonstrated regenerative potential in many preclinical studies. However, traditional techniques for isolating MSCs typically involve a bone marrow harvest from the iliac crest. Due to the invasive nature of the iliac crest bone marrow harvest procedure and the developmental (endochondral) origin of this tissue, it would be far more desirable to have a more readily accessible and developmentally similar (intramembranous) source of MSCs for craniofacial therapeutic indications. To this end, we have preliminary evidence which suggests that MSCs can be predictably isolated from alveolar bone marrow, using a simple, standardized, reproducible technique. Our hypothesis is that using current Good Manufacturing Practices (cGMP) guidelines, alveolar bone marrow derived MSCs (aBMSCs) can be safely expanded ex vivo, to numbers sufficient for autogenous clinical transplantation in human craniofacial bone defects. Additionally, we hypothesize that in vitro and preclinical surrogate markers exist which can be predictive of clinical regenerative outcomes. To test these hypotheses, the project proposed has three Specific Aims (SAs). In SA1, we will isolate, expand, and phenotypically characterize populations of alveolar bone marrow derived stem cells from 30 different human subjects. SA2 will evaluate the preclinical bone regenerative potential of aBMSCs in vivo using ectopic and clinically relevant orthotopic model systems. Finally, in SA3, following autologous transplantation of aBMSCs into human craniofacial bone defects, we will determine how the phenotypic characteristics of different aBMSC populations correlate to their respective capacities to regenerate bone in vivo and clinically. Key outcome measures include: phenotypic characterization of aBMSCs; in vivo potential of aBMSCs to regenerate bone and vascular tissue; safety of aBMSC cell isolation and expansion methodologies for human application; clinical potential of aBMSCs to regenerate bone and vascular tissue; and examination of correlations between in vitro phenotype, in vivo bone regeneration, and clinical bone regeneration. Findings of the proposed study could significantly advance clinical cell therapy approaches for craniofacial regeneration. Additionally, the isolation of aBMSCs with the technique employed would have broad scientific impact in providing an alternative source of MSCs to be used in fundamental investigations of stem cell and bone biology.